Context: Management of wounds is frequently encountered with different problems. Drug resistance and toxicity have hindered the development of synthetic antimicrobial agents with wound healing activity. Many plants with multi-potent pharmacological activities may offer better treatment options. Triphala (dried fruits of Terminalia chebula, Terminalia bellirica, and Phyllanthus emblica) formulations have shown healing activity on wounds. Aims: Aim was to compare the wound healing activity of jethimadh with triphala in rats. Materials and Methods: An ointment was prepared by mixing Jethimadh (Glycyrrhizae glabra Linn) powder in ghee obtained from cow's milk.Triphala ointment was prepared by mixing triphala powder in sesame oil. Both jethimadh and triphala were evaluated for healing activity on rat model of incised and excised wounds. Parameters evaluated for assessment of efficacy of the formulations are tensile strength, level of hydroxyproline, and wound contraction. Results: Efficacy was significant (P < 0.001) with both jethimadh and triphala groups compared with control. Efficacy of Jethimadh is significantly higher compared with triphala in incision wound model, but no statistically significant difference was observed between these two groups in excision wound model. Conclusion: The data suggest that healing activity of jethimadh with respect to triphala is significantly higher on rat model of incised wound and is comparable on rat model of excised wound.

Animal wound healing models are important biological tools to understand basic processes of tissue repair and to develop and validate strategies for treatment of wounds. Wound healing in human beings has many unique aspects that relate to physiology, age, environmental factors, etc., but the opportunities to carry out clinical experiments to understand mechanism and to formulate therapy for wound healing are limited. Although animal wound healing models are imperfect reflection of wound healing processes in human beings and its clinical challenges, these models continue to be crucial tools for the development of new strategies and approaches for therapy of wound healing. [1]

Wound healing is a complex dynamic process which involves an array of interrelated and concomitant events. The process of wound healing differs little from one kind of tissue to another and is generally independent of the form of injury. The phases of wound healing are hemostasis, epithelization, formation of granulation tissue, and remodeling of extracellular matrix. Management of wounds is frequently encountered with different problems. Drug resistance and toxicity have hindered the development of synthetic antimicrobial agents with wound healing activity. Many plants with multi-potent pharmacological activities may offer better treatment options. Triphala (dried fruits of Terminalia chebula, Terminalia bellirica, and Phyllanthus emblica) formulations have shown healing activity on wound models in rats. [2]

Jethimadh is a traditional ayurvedic herbal formulation consisting of the dried roots and rhizomes of Jethimadh (Glycyrrhizae glabra Linn). The major constituents of jethimadh are triterpene saponins. Glycyrrhizin (glycyrrhizic acid, glycyrrhizinic acid) is the active principle of jethimadh. [3] Dried roots and rhizomes of jethimadh have shown anti-inflammatory, antiulcer, and antioxidant activity. [4] Based on these properties of jethimadh, a pilot study was conducted by the authors to assess its healing activity on wounds in rats. In that study, Jethimadh showed promising outcomes in the management of wounds. Against this backdrop, Jethimadh was included as a study formulation in order to compare its healing activity with triphala on rat model of incised and excised wounds. Parameters evaluated for assessment of efficacy of the formulations are tensile strength and level of hydroxyproline (estimation of collagen formation) in incision wound model and wound contraction in excision wound model.

Experimentally naive male Sprague Dawley albino rats weighing between 150 and 200 g were used. The rats were maintained under standard conditions of temperature (25°C ± 5°C), relative humidity (55 ± 10%), and a 12/12- hour light/dark cycle. The rats were fed with commercial rat pellet diet manufactured by Pranav Agro Food, Pune, and water ad libitum. The rats were procured from institutional animal house (Registration no-619/02/a/CPCSEA) and the study was approved by the Institutional Animal Ethics Committee.

Incision wound model

The anesthetized (ketamine - 30 mg/kg i.p.) animals were secured to the operation table in prone position. [5] Two paravertebral straight line incisions of 6 cm each were made as described by Ehrlich et al. 2001. [6] Care was taken to see that incisions were at least 1 cm lateral to the vertebral column. After complete hemostasis, wounds were closed by interrupted sutures. Thereafter, rats were divided into five groups. Group I (n = 10) ─ Sesame oil group, served as control for triphala group. Group II (n = 10) ─ Cow's ghee group served as control for Jethimadh group. Group III (n = 10) ─ Framycetin group, served as positive control. Group IV (n = 10) ─ Triphala group, the comparator group. Group V (n = 10) ─ Jethimadh group, the study group. The formulations were applied topically with a sterile cotton swab in rats of each group once daily. Stitches were removed on day 8 and treatment was continued. [5],[6]

Evaluation

Evaluation was done by measuring tensile strength using a Tensiometer. [5] It was measured on day 10 under anesthesia (ketamine - 30 mg/kg i.p.) by the continuous, constant water flow technique of Lee 1968 [Figure 1]. [7]

Collagen deposition in the wound was measured by estimating hydroxyproline in tissue. The healed tissue was obtained on the day 10 from five study groups to estimate hydroxyproline levels as described by Neuman and Logan 1950. [8]

Excision wound model

The anesthetized (ketamine - 30 mg/kg) animals were secured to the operation table in prone position. An impression was made on dorsal part of thoracic region. The skin was excised to obtain a full thickness wound of 500 mm 2[Figure 2]. Hemostasis was secured with cotton swabs soaked in normal saline solution. Rats were divided into five groups of ten rats each as done in the previous model. Study formulations were applied topically with a sterile cotton swab in rats of each group once daily till wounds were healed completely. Wound contraction was studied by tracing the wound area on a transparent sheet on day 0, 3, 6, 9, 12 and subsequently measuring wound area on alternate days till complete epithelization. Complete epithelization was identified by the fall of scab without any raw area. [9]

Tensile strength and level of hydroxyproline in different groups were expressed as mean ± SD. Contraction of wound was expressed as percentage of wound closure. Data were analyzed by ANOVA followed by Turkey's honestly significant different test (HSD). The criterion for statistical significance was defined as P<0.05.

Results

Tensile strength

Tensile strength in different groups has been shown in [Figure 3]. Tensile strength in group IV 427 ± 11.45 g (mean ± SD) and group V 517 ± 11.09 g were significantly higher compared with group I (322 ± 11.24 g), group II (372 ± 10.1 g), and group III (234 ± 7.69 g). Tensile strength in group V was significantly higher compared with Group IV.

Complete wound closure was observed in Triphala ointment-treated group on day 21. So, the percentages of wound closure in different groups on day 21 were compared and have been shown in [Figure 5]. Mean percentage of wound closure in group IV (100%) was significantly higher compared with group I (93.16%) and also in group V (98.72%) compared with group II (92.36 %). There is no significant difference of percentage of wound closure among groups III, IV, and V.

Wound healing is a fundamental response to tissue injury that results in restoration of tissue integrity. Currently, the treatment options for majority of wounds include more of supportive measures (maintenance of hygiene, proper dressing, and prophylactic use of antibiotics) rather than agents that actually enhance the process of wound healing. The present study was undertaken based on information obtained from Ayurveda system of medicine about certain herbal preparations such as triphala, honey, etc., which have inherent wound healing properties. Thereafter, the study formulation jethimadh was compared with triphala for its healing activity on wounds in rats.

Our study revealed that formulation of jethimadh in cow ghee and triphala in sesame oil have shown great promise in promoting wound healing. The above data suggest that healing activity was significant (P<0.001) in both jethimadh and triphala-treated groups. Healing activity of jethimadh with respect to triphala is significantly higher on rat model of incised wound and is comparable on rat model of excised wound. Tensile strength of the wound reflects ability of the healed tissue to sustain maximum load. Increased tensile strength in incision wound model suggests that jethimadh increases number of fibroblasts and rate of collagen synthesis in the treatment group. [10] Increased hydroxyproline level in incision wound model suggest that jethimadh induces increased fibroblastic proliferation and thereby increased collagen synthesis. [11] Collagen confers strength and integrity to the tissue matrix and plays an important role in homeostasis and epithelization. Early wound contraction in excision wound model suggests that triphala and jethimadh have positive effect toward cellular proliferation, granulation tissue formation, and epithelization. [12]

Wound contraction occurs through the centripetal movement of the tissues surrounding the wound, which is mediated by myofibroblasts. [13] Increased wound contraction in the triphala and jethimadh-treated groups may be due to the enhanced activity of fibroblasts and successful elimination of bacteria by triphala and jethimadh. The slow rate of wound closure in the control groups (groups I and II) might be attributed to the presence of microorganisms and their metabolites, which inhibit wound contraction and impair healing. Significant increase in collagen content during the wound healing process in the triphala and jethimadh-treated groups resulted due to enhanced migration of fibroblasts and epithelial cells to the healing tissue. [2] Moreover, Triphala contains ascorbic acid, which is a cofactor for the synthesis of collagen as well as elastin fibers. [14] The decreased collagen content in the control groups might be due to prolonged inflammatory phase where the degradation of collagen is more than the synthesis. During the early inflammatory phase of wound healing, polymorphonuclear leukocytes and macrophages infiltrate the healing tissue and produce large amount of reactive oxygen species (ROS) as part of their defense mechanism. Although this process is beneficial, increased levels of ROS can inhibit cell migration and proliferation and can even cause severe tissue damage. [2] The presence of free radicals result in oxidative stress leading to lipid peroxidation, DNA breakage, and enzyme inactivation, including free radical scavenger enzymes. Proteins are the main target sites for the generation of free radicals in tissue, and antioxidants that are capable of scavenging the free radicals can be used to minimize the damage to the proteins. [15],[16] Triphala scavenges the free radicals effectively and decreases the oxidative stress. [17] This antioxidant activity of Triphala helps to inhibit ROS on wounds and enhance healing. [2] Similarly, antioxidant activity of jethimadh may be responsible for would healing activity of jethimadh.

In conclusion, the above data suggest that the healing activity of Jethimadh with respect to triphala is significantly higher on rat model of incised wound and is comparable on rat model of excised wound. Further phytochemical studies are required where the jethimadh extract will be subjected to further fractionation and purification to identify the active principles responsible for their pharmacological activities and their mechanisms toward wound healing. This knowledge will further enable us to carry out extensive clinical trials of these formulations on human volunteers so as to provide better medications for wound healing in future.